Studies on the Arabidopsis LINE, ATLNs : Their expression and the function of ORF proteins encoded.

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The Arabidopsis thaliana genome has LINEs, called ATLNs, whose expression is negatively regulated by DNA methylation. Analysis of deletion in the 5' UTR revealed that the 5' UTR has two promoters, designated here as P1 and P2. Analysis of transcripts by 5' RACE showed that their 5' ends were located at sites immediately upstream of the P1 region or at sites downstream of the P2 region. This observation and the fact that the P1 region contains no TATA sequence indicate that P1 is an internal promoter that initiates transcription from sites upstream of the promoter.ATLN members have two consecutive open reading frames (orf1 and orf2) that are essential for retrotransposition. The C-terminal region of the ORF1 protein has nuclear localization signal (NLS) -like sequences, KRKR and KKGRK. The GFP-ORF1 fusion protein, which was expressed in tobacco BY-2 and onion skin cells, was localized to the nuclei of their cells, but the GFP-ORF1-fusion protein with mutations in NLS-like sequences was not. Because the N-terminal region of the ORF1 protein has a retroviral zinc-finger motif, we also examined protein-protein interactions by using two hybrid system in yeast as well as in tobacco BY2 cells. We found that ORF1 can interact with each other, whereas ORF1 with substitution mutations in the zinc-finger motif as well as in the leucine-zipper motif cannot. These results show that ORF1 consists of two kinds of functional domains involved in nuclear localization and protein-protein interaction.